Application of engineered exosomes in the preparation of products that improve the survival rate of prefabricated skin flaps

Abstract

The present invention discloses an application of engineered exosomes in the preparation of a product for improving the survival rate of pre-constructed skin flaps. The engineered exosomes are prepared by the following method: VEGF plasmids are introduced into adipose-derived stem cells by electroporation technology and incubated, and the exosomes are then collected by differential centrifugation to obtain the engineered exosomes. The present invention introduces VEGF plasmids into adipose-derived stem cells and then obtains engineered exosomes by incubation and separation, which can improve the functional substances in the exosomes, so that they can better promote the blood supply reconstruction of the flap and significantly improve the survival rate of the pre-constructed skin flap. In addition, by loading the engineered exosomes into tannic acid hydrogels, a sustained release effect on the exosomes can be achieved and they have certain antibacterial properties, effectively avoiding the problems of poor targeting of exosomes, easy clearance from the circulation, short half-life, and the need for multiple injections.

Description

Technical Field

[0001] The present invention relates to the technical field of prefabricated skin flap repair, and in particular to the use of engineered exosomes in preparing a product for improving the survival rate of prefabricated skin flaps. Background Art

[0002] A prefabricated flap is a surgical procedure in which tissues such as the aponeurosis, muscle, or omentum, which contain known blood vessels, are transplanted to a specific layer (usually the subcutaneous tissue layer) in an area (or region) that originally lacks known blood vessels. Alternatively, a free skin graft is transplanted onto tissues such as the aponeurosis, muscle, or omentum, which have known vascular bundles. After a period of revascularization, a new axial flap, supplied by known vascular bundles, is formed, which can then be used for secondary surgical repair of the defect with a pedicle or free graft. The advent of prefabricated flaps transcends the limitations of traditional flap selection based on the distribution of known surface blood vessels, broadens the donor area during surgery, and significantly increases the number of flap options. Prefabricated flaps also feature controllable thickness, smoothness, and a large surface area, thus avoiding bulk when repairing delicate areas and resulting in a more aesthetically pleasing result. In recent years, they have become a popular technique for repairing complex head and facial defects.

[0003] While prefabricated flaps possess the aforementioned advantages, they also suffer from a significant drawback: a high rate of partial necrosis after surgery, limiting their widespread clinical application. Inadequate flap revascularization after primary surgery can easily lead to flap necrosis after secondary transplantation. Therefore, improving the survival rate of prefabricated flaps is a crucial area of ​​research in reparative and reconstructive surgery.

[0004] At present, in addition to the use of extended surgical intervals and flap delay surgery, as well as routine perioperative measures such as antispasmodics, warmth preservation, anticoagulation, and microcirculation promotion, there is no reliable means to improve the survival rate of prefabricated flaps. Partial necrosis or even failure of prefabricated flaps remains a difficult problem faced in clinical practice.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of engineered exosomes in the preparation of a product for improving the survival rate of prefabricated skin flaps. The engineered exosomes of the present invention can promote the revascularization of the skin flap and significantly improve the survival rate of the prefabricated skin flap.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] A first aspect of the present invention provides a use of engineered exosomes in preparing a product for improving the survival rate of a prefabricated skin flap, wherein the engineered exosomes are prepared by the following method:

[0009] The VEGF plasmid is introduced into adipose-derived stem cells by electroporation technology and incubated, and then the exosomes are collected by differential centrifugation to obtain the engineered exosomes.

[0010] Preferably, the use of the engineered exosomes combined with ovalbumin polypeptide in the preparation of a product for improving the survival rate of prefabricated skin flaps;

[0011] The ovalbumin polypeptide is prepared by enzymatic hydrolysis of ovalbumin with trypsin and papain. Preferably, the VEGF plasmid is obtained by inserting a gene expressing VEGF protein into a vector.

[0012] Preferably, the incubation time is 10 to 14 hours.

[0013] A second aspect of the present invention provides a method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, the method comprising the following steps:

[0014] (a) GelMA, the engineered exosomes, and an initiator solution are mixed and heated to dissolve in the dark, and then irradiated with a 405 nm light source to obtain an exosome-loaded gel;

[0015] (b) The exosome-loaded gel is immersed in a tannic acid solution to obtain the hydrogel for improving the survival rate of the prefabricated skin flap.

[0016] Preferably, in step (a), the initiator is LAP, and the concentration of the initiator solution is 0.2% to 0.3%.

[0017] Preferably, in step (a), the mass volume ratio of GelMA to initiator solution is 1:(18-22);

[0018] The mass-to-volume ratio of engineered exosomes to initiator solution was 1:(2000-4000);

[0019] The heating solvent temperature is 60~70℃ and the time is 20~30min.

[0020] Preferably, the step (a) further comprises adding ovalbumin polypeptide and mixing before irradiation;

[0021] The mass volume ratio of ovalbumin polypeptide to initiator solution is 1: (800~1200).

[0022] Preferably, in step (b), the concentration of the tannic acid solution is 8% to 12%.

[0023] Preferably, in step (b), the volume ratio of the exosome-loaded gel to the tannic acid solution is 1:(2-3), and the soaking time is 4-8 hours.

[0024] A third aspect of the present invention provides a hydrogel prepared by the above preparation method for improving the survival rate of prefabricated skin flaps.

[0025] Compared with the prior art, the beneficial effects of the present invention include at least:

[0026] The present invention introduces VEGF plasmid into adipose stem cells and then incubates and separates them to obtain engineered exosomes, which can improve the functional substances in the exosomes so that they can better promote the blood circulation reconstruction of the skin flap, significantly improve the survival rate of the pre-constructed skin flap, and significantly reduce the contracture of the skin flap; in addition, by combining the engineered exosomes with ovalbumin polypeptides, it can better promote the blood circulation reconstruction of the skin flap and significantly improve the survival rate of the pre-constructed skin flap. In addition, the engineered exosomes and ovalbumin polypeptides are loaded into tannic acid hydrogels, which can achieve a sustained release effect on the exosomes and have certain antibacterial properties, effectively avoiding the problems of poor targeting of exosomes, easy clearance from the circulation, short half-life, and the need for multiple injections. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0028] Figure 1 This is a transmission electron micrograph of the engineered exosomes in the experimental example of the present invention;

[0029] Figure 2 is a scanning electron micrograph of the tannic acid hydrogel in the experimental example of the present invention;

[0030] Figure 3 The antibacterial effects of PBS, gel and tannic acid hydrogel in the experimental examples of the present invention are shown;

[0031] Figure 4 The fluorescence of the skin flap is observed by an in vivo imaging device in the experimental example of the present invention;

[0032] Figure 5 These are observation pictures of skin flaps in different treatment groups at different times after in situ suture in the experimental example of the present invention;

[0033] Figure 6 This is a statistical diagram of flap survival in different treatment groups at different times after in situ suturing in the experimental example of the present invention;

[0034] Figure 7 The wound contracture conditions of different treatment groups in the experimental examples of the present invention;

[0035] Figure 8Figure 2 is the result of immunohistochemistry of vWF in different treatment groups in the experimental examples of the present invention;

[0036] Figure 9 Statistical analysis of the vWF immunohistochemistry detection results of different treatment groups in the experimental examples of the present invention. DETAILED DESCRIPTION

[0037] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0038] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0039] An embodiment of the present invention provides a use of engineered exosomes in preparing a product for improving the survival rate of a prefabricated skin flap, wherein the engineered exosomes are prepared by the following method:

[0040] The VEGF plasmid is introduced into adipose-derived stem cells by electroporation technology and incubated, and then the exosomes are collected by differential centrifugation to obtain the engineered exosomes.

[0041] The present invention introduces VEGF plasmids into adipose-derived stem cells and then obtains engineered exosomes through incubation and separation, which can improve the functional substances in the exosomes, enable them to better promote the revascularization of the flap, and significantly improve the survival rate of the pre-constructed flap.

[0042] In one embodiment, the engineered exosomes are produced by the following method:

[0043] Adipose-derived stem cells were seeded on the upper layer of a 1cm×1cm cell nanoporous silicon chip and incubated overnight. A VEGF plasmid suspended in PBS solution was injected into the lower layer of the cell nanoporous silicon chip at a concentration of 100ng / ml. The instrument parameters were set to 80V voltage, 3ms pulse width, 100ms interval, and 100 repetitions. Then, current was applied and the plasmid in PBS buffer was injected into the upper layer of cells through nanochannels (diameter approximately 500nm, spacing 5µm). The treated cells were then incubated for 10-14h, and the upper layer of the chip was collected, and the engineered exosomes were extracted by differential centrifugation.

[0044]

[0045] In one embodiment, the use of engineered exosomes combined with ovalbumin polypeptide in the preparation of a product that improves the survival rate of prefabricated skin flaps;

[0046] The ovalbumin polypeptide is prepared by enzymatic hydrolysis of ovalbumin with trypsin and papain.

[0047] The present invention uses engineered exosomes in combination with ovalbumin polypeptide to better promote the revascularization of the flap and significantly improve the survival rate of the pre-fabricated flap.

[0048] Another embodiment of the present invention provides a method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, the method comprising the following steps:

[0049] (a) GelMA, the engineered exosomes, and an initiator solution were mixed and heated to dissolve in the dark, and then irradiated with a 405 nm light source to obtain an exosome-loaded gel;

[0050] (b) The exosome-loaded gel is immersed in a tannic acid solution to obtain the hydrogel for improving the survival rate of the prefabricated skin flap.

[0051] By loading engineered exosomes into tannic acid hydrogel, the present invention can achieve a sustained release of exosomes and have certain antibacterial properties, effectively avoiding the problems of poor targeting of exosomes, easy clearance from the circulation, short half-life, and the need for multiple injections, and can significantly improve the survival rate of pre-constructed skin flaps.

[0052] In one embodiment, in step (a), the initiator is LAP, and the concentration of the initiator solution can be any value between 0.2% and 0.3%, specifically 0.2%, 0.25% or 0.3%.

[0053] In one embodiment, in step (a), the mass volume ratio of GelMA to the initiator solution can be any value in the range of 1: (18-22), specifically 1:18, 1:20 or 1:22;

[0054] The mass-to-volume ratio of the engineered exosomes to the initiator solution can be any value between 1: (2000-4000).

[0055] In one embodiment, in step (a), the temperature of the heated solvent may be any value between 60° C. and 70° C., and the time may be any value between 20 and 30 minutes.

[0056] In one embodiment, the step (a) further comprises adding ovalbumin polypeptide and mixing before irradiation;

[0057] The mass volume ratio of ovalbumin polypeptide to initiator solution is 1: (800~1200).

[0058] In one embodiment, in step (b), the concentration of the tannic acid solution may be any value between 8% and 12%.

[0059] In one embodiment, in step (b), the volume ratio of the exosome-loaded gel to the tannic acid solution can be 1:(2-3), and the soaking time can be 4-8 hours.

[0060] Yet another embodiment of the present invention provides a hydrogel prepared by the above preparation method for improving the survival rate of prefabricated skin flaps.

[0061] The technical solution of the present invention is further described in detail below through specific embodiments.

[0062] The raw materials used in the following examples are as follows:

[0063] Ovalbumin polypeptide was prepared by the following method:

[0064] Place 0.5 g of ovalbumin (Isejiu Biotechnology 9006-59-1) in 10 ml of distilled water, add 0.5 ml of trypsin digestion solution (1%, Isejiu Biotechnology), and enzymatically hydrolyze at 37°C for 2 hours. Then, add 5 mg of papain (≥1 million u / g, Isejiu Biotechnology) and enzymatically hydrolyze at 50°C for 2 hours. After enzyme inactivation, ultrafiltration is performed to retain polypeptides with a molecular weight cutoff of 500-1000 Da, and freeze-drying is performed to obtain ovalbumin polypeptide.

[0065] Example 1

[0066] This embodiment provides a method for preparing engineered exosomes, which comprises the following steps:

[0067] 2×10 5 Adipose-derived stem cells were seeded on the upper layer of a 1cm×1cm cell nanoporous silicon chip and incubated overnight. A VEGF plasmid suspended in PBS solution was injected into the lower layer of the cell nanoporous silicon chip at a concentration of 100ng / ml. The instrument parameters were set to 80V voltage, 3ms pulse width, 100ms interval, and 100 repetitions. Then, current was loaded and the plasmid in PBS buffer was injected into the upper cells through nanochannels (diameter approximately 500nm, spacing 5µm). The treated cells were then incubated for another 12h, and the upper liquid of the chip was collected. The engineered exosomes (denoted as VEGF-EV) were extracted by differential centrifugation.

[0068] Wherein, VEGF plasmid was prepared by the following method:

[0069] The gene expressing VEGF protein (its sequence is shown in SEQ ID NO: 1) is inserted into the vector to obtain the VEGF plasmid structure. (Commissioned by Yunzhou Biotechnology (Guangzhou) Co., Ltd. for synthesis).

[0070] Example 2

[0071] This embodiment provides a method for preparing a tannic acid hydrogel, which comprises the following steps:

[0072] Place 100 mg of GelMA in a centrifuge tube. Add 2 ml of initiator solution (0.25% LAP) to the tube and shake to fully soak the GelMA.

[0073] Heat and dissolve in a 65°C water bath in the dark for 25 min, shaking three times during the process to mix thoroughly; irradiate with a 405 nm light source for 15 seconds to gel, obtaining a gel (denoted as Gel);

[0074] Prepare 5 ml of 10% tannic acid solution, and soak the above gel in the tannic acid solution for 6 h to obtain tannic acid hydrogel (denoted as TA-Gel).

[0075] Example 3

[0076] This embodiment provides a method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, and the preparation method comprises the following steps:

[0077] 100 mg of GelMA and 0.56 mg of the engineered exosomes of Example 1 were placed in a centrifuge tube. 2 ml of the initiator solution (0.25% LAP) was added to the tube and shaken to allow the GelMA to fully infiltrate.

[0078] Heat and dissolve in a 65°C water bath in the dark for 25 min, shaking three times to mix thoroughly; irradiate with a 405 nm light source for 15 seconds to gel, obtaining an exosome-loaded gel;

[0079] 5 ml of 10% tannic acid solution was prepared, and the exosome-loaded gel was immersed in the tannic acid solution for 6 h to obtain a hydrogel that improved the survival rate of the prefabricated skin flap (denoted as VEGF-EV@TA-Gel).

[0080] Example 4

[0081] This embodiment provides a method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, and the preparation method comprises the following steps:

[0082] 100 mg of GelMA and 0.56 mg of the engineered exosomes of Example 1 were placed in a centrifuge tube. 2 ml of the initiator solution (0.25% LAP) was added to the tube and shaken to allow the GelMA to fully infiltrate.

[0083] Heat and dissolve in a 65°C water bath in the dark for 25 minutes, shaking three times during the process to mix thoroughly. Then, add 0.56 mg of ovalbumin polypeptide and mix thoroughly. Irradiate with a 405 nm light source for 15 seconds to gel, thereby obtaining an exosome-loaded gel.

[0084] 5 ml of 10% tannic acid solution was prepared, and the exosome-loaded gel was immersed in the tannic acid solution for 6 h to obtain a hydrogel that improved the survival rate of the prefabricated skin flap (denoted as VEGF-EV-PP@TA-Gel).

[0085] Comparative Example 1

[0086] This comparative example is a method for preparing exosomes, which comprises the following steps:

[0087] 2×10 5 Adipose-derived stem cells were seeded on the upper layer of a 1cm×1cm cell nanoporous silicon chip and incubated overnight. PBS solution was injected into the lower layer of the cell nanoporous silicon chip. The instrument parameters were set to 80V voltage, 3ms pulse width, 100ms interval, and 100 repetitions. The treated cells were then incubated for 12 hours, and the liquid on the chip was collected, and the exosomes (referred to as empty EVs) were extracted by differential centrifugation.

[0088] Comparative Example 2

[0089] This comparative example is a method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, and the preparation method comprises the following steps:

[0090] Place 100 mg of GelMA in a centrifuge tube. Add 2 ml of initiator solution (0.25% LAP) to the tube and shake to fully soak the GelMA.

[0091] Heat and dissolve in a 65°C water bath in the dark for 25 minutes, shaking three times during the process to mix thoroughly; then add 0.56 mg of ovalbumin polypeptide and mix thoroughly, and irradiate with a 405 nm light source for 15 seconds to gel, thereby obtaining a polypeptide-loaded gel;

[0092] Prepare 5 ml of 10% tannic acid solution, and soak the above-mentioned peptide-loaded gel in the tannic acid solution for 6 hours to obtain a hydrogel that improves the survival rate of the prefabricated skin flap (denoted as Polypeptide@TA-Gel or PP@TA-Gel).

[0093] Experimental example

[0094] 1. The engineered exosomes in Example 1 and the empty EVs in Comparative Example 1 were dropped onto a copper mesh, then stained with phosphotungstic acid for 30 seconds. The dye was removed by aspiration, and the mesh was washed twice with ultrapure water. The mesh was then photographed under a transmission electron microscope. The results are shown in the figure. Figure 1 As shown;

[0095] Depend on Figure 1 It can be seen that:

[0096] Figure 1 The left picture in the middle is empty EV, and the right picture is VEGF-EV. Both have typical cup-shaped structures and smooth double-layer structures, indicating that CNP has no effect on the size and shape of EV.

[0097] 2. The tannic acid hydrogel prepared in Example 2 was freeze-dried for 18 h, then sliced ​​and photographed using a scanning electron microscope. The results are as follows: Figure 2 As shown;

[0098] Depend on Figure 2 It can be seen that:

[0099] The surface morphology of the TA-Gel scaffold is a smooth, continuous porous three-dimensional network structure, which is conducive to the loading of VEGF-EV.

[0100] 3. Soak 1 ml of 10% Gel and 10% TA-Gel in 2 ml of PBS solution for 4 hours, then co-culture the PBS and the two soaking solutions with Escherichia coli and Staphylococcus aureus in the logarithmic growth phase for 8 hours;

[0101] The two bacterial suspensions after treatment were stained with SYTO-9 and PI for live and dead staining, and photographed using a confocal microscope. The photographic results are shown in Figure 2. Figure 3 As shown;

[0102] Depend on Figure 3 It can be seen that:

[0103] Both detection methods showed that the solution soaked in TA-Gel could significantly inhibit bacterial growth after incubation with bacteria, indicating that TA-Gel has an antibacterial effect, which can prevent bacterial infection during the revascularization process and accelerate the revascularization process.

[0104] 4. Animal experiments on prefabricated skin flaps:

[0105] Surgical Plan: The rat prefabricated flap model was constructed in two stages. In the first stage, adult Sprague-Dawley rats weighing approximately 280g were obtained. A rectangular flap, approximately 4 x 4.5 cm, was created on the abdomen, extending from the xiphoid process to 1 cm above the external genitalia and to the anterior axillary line on either side. The flap was raised from the superior to the inferior aspect, and the superficial epigastric arteries were ligated on both sides. The femoral artery and vein bundle of the right lower extremity was freed, preserving a small amount of surrounding muscle tissue, and then the branch vessels were ligated. The bundle was then transposed subcutaneously through the groin at the ankle joint to the inner surface of the flap. The vascular bundle was secured to the inner surface of the flap with 6-0 sutures. After hemostasis, the wound was sutured in situ.

[0106] Ten days later, the second stage of surgery was performed. ICG was injected into the tail vein before the surgery. The island flap was then completely freed along the original incision, leaving only the implanted axial vascular bundle as the pedicle. The fluorescence of the flap was then observed using an animal in vivo imaging device. The results were as follows: Figure 4 As shown, Figure 4 The upper left side of the middle image is the untreated normal rat skin control group;

[0107] Depend on Figure 4 It can be seen that:

[0108] The flap showed a fan-shaped fluorescence distribution centered on the vascular pedicle, indicating that the vascular pedicle was unobstructed and supplied blood to the flap.

[0109] Ten days after surgery, a second-stage surgery was performed. After raising an island flap pedicled with the implanted axial vascular bundle along the original incision, the flap was given different treatments (Control group: 3.6 ml PBS, applied evenly; VEGF-EV group: 3.6 ml PBS containing 1 mg VEGF-EV, applied evenly; TA-Gel group: 3.6 ml TA-Gel, covering the flap position; VEGF-EV@TA-Gel group: 3.6 ml TA-Gel loaded with 1 mg VEGF-EV, covering the flap position; PP@TA-Gel group: 3.6 ml TA-Gel loaded with 1 mg Polypeptide, covering the flap position; VEGF-EV-PP@TA-Gel group: 3.6 ml TA-Gel loaded with 1 mg VEGF-EV and 1 mg Polypeptide, covering the flap position), and then the flap was sutured in situ. The survival of the flap was observed two weeks after surgery.

[0110] Observation results such as Figure 5 The statistical analysis results are shown in Figure 6 As shown by Figure 5 、 Figure 6 It can be seen that:

[0111] The flap survival areas of the first four groups were observed on the 1st, 4th and 7th days after the second stage surgery. It was found that the flap survival area of ​​the VEGF-EV@TA-Gel group was the largest. After statistical analysis, the difference was found to be statistically significant.

[0112] The contracture of the first four groups of wounds was further observed and statistically analyzed. Figure 7 As shown;

[0113] Depend on Figure 7 It can be seen that:

[0114] The flap in the VEGF-EV@TA-Gel group had only slight contracture, while the first three groups all had severe contracture, and the contracture in the first group was the most severe, and the difference was statistically significant.

[0115] The skin flap tissues of the upper right corner of the six groups of rats were subjected to immunohistochemical detection of vWF, and the vWF content was statistically analyzed.

[0116] Immunohistochemistry results Figure 8 The statistical analysis results are shown in Figure 9 As shown by Figure 8 、 Figure 9 It can be seen that:

[0117] The vWF content in the VEGF-EV@TA-Gel group, Polypeptide@TA-Gel group, and VEGF-EV-PP@TA-Gel group increased to varying degrees, and the vWF content in the VEGF-EV-PP@TA-Gel group was the highest, followed by the VEGF-EV@TA-Gel group and Polypeptide@TA-Gel group. The differences were statistically significant.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for preparing a hydrogel for improving the survival rate of a prefabricated skin flap, characterized in that: The preparation method comprises the following steps: (a) GelMA, engineered exosomes, and an initiator solution are mixed and heated to dissolve in the dark, and ovalbumin polypeptide is added and mixed thoroughly; the mixture is then irradiated with a 405 nm light source to obtain an exosome-loaded gel; the engineered exosomes are prepared by introducing a VEGF plasmid into adipose-derived stem cells via electroporation and incubating the cells, followed by collection of the exosomes by differential centrifugation; the VEGF plasmid is obtained by inserting a gene expressing the VEGF protein into a vector; and the incubation time is 10 to 14 hours. The ovalbumin polypeptide is prepared by enzymatically hydrolyzing and inactivating ovalbumin with trypsin and papain, ultrafiltration to retain polypeptides with a molecular weight cutoff of 500-1000 Da, and freeze-drying; the mass-to-volume ratio of the ovalbumin polypeptide to the initiator solution is 1:(800-1200); (b) soaking the exosome-loaded gel in a tannic acid solution to obtain the hydrogel for improving the survival rate of the prefabricated skin flap; In step (a), the initiator is LAP, the concentration of the initiator solution is 0.2% to 0.3%; the mass volume ratio of GelMA to the initiator solution is 1:(18-22); the mass volume ratio of the engineered exosomes to the initiator solution is 1:(2000-4000); the heating and dissolving temperature is 60-70°C, and the time is 20-30 minutes; In step (b), the concentration of the tannic acid solution is 8% to 12%; the volume ratio of the exosome-loaded gel to the tannic acid solution is 1:(2 to 3), and the soaking time is 4 to 8 hours.

2. A hydrogel for improving the survival rate of prefabricated skin flaps prepared by the preparation method of claim 1.

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